Human Cytomegalovirus Infection in Endothelial Cells Induces the Secretory Autophagy Pathway for Egress
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PublisherThe University of Arizona.
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AbstractHuman cytomegalovirus (HCMV) is a β-herpesvirus that establishes a lifelong infectionwith the potential to infect multiple cell types, including vascular endothelial cells. While infection in fibroblasts is well-characterized, much remains to be understood about key differences in the infection of other cell types. Endothelial cells support a slow and chronic infection while contributing to the spread of the virus to distal organs. However, the cellular and molecular determinants of HCMV infection in endothelial cells remain poorly understood. Both infected endothelial cells and fibroblasts incorporate viral material, like virions and dense bodies, into a multivesicular body (MVB)-like vesicle. However, these vesicles have distinct biogenesis in each cell type. Virus-containing vesicles in fibroblasts are classical MVBs marked by CD63 and LBPA. However, characterization of the vesicles in endothelial cells showed that cis-Golgi (GM130), lysosomal-associated membrane protein- 1 (LAMP1) proteins are on the limiting membrane of the virus-induced vesicles, and autophagy maker LC3B is on the membranes of intraluminal vesicles. These markers are commonly associated with the secretory autophagy pathway. Moreover, the vesicles in endothelial cells lack CD63 and LBPA. These results suggest that the virus-containing vesicles in infected endothelial cells may traffic through the secretory autophagy pathway. Our findings support this suggestion in that we have found differences in protein levels of Rab8A and Rab8B, two proteins that distinguish degradative autophagy from secretory autophagy, in infected endothelial cells. These findings suggest that HCMV induces the secretory autophagy pathway in endothelial cells possibly for egress. Furthermore, HCMV has evolved mechanisms to co-opt host trafficking pathways for transport, virion maturation, and egress among other functions. The viral protein UL135 has been shown to have a role in the rearrangement of membrane trafficking, and incorporation of virus material into MVB-like vesicles. We investigated the role of UL135 in the biogenesis of the virus-induced vesicles and its involvement in the induction of the secretory autophagy pathway during infection using immunoblotting for both viral proteins and well-characterized markers of secretory and degradative autophagy. We found that, in the absence of pUL135, Rab8A, LC3B, and P62 are increased, suggesting that pUL135 is involved in the decrease of lysosomal activity thus the decrease in degradation. This may suggest that UL135 may be involved in the biogenesis of the LAMP-1, GM130, LC3B positive vesicles and possibly give preference to the secretory autophagy pathway when incorporating virus material into the vesicles for egress. This work provides novel insights into the diversity of mechanisms by which HCMV hijacks membrane trafficking in different cell types and will reveal cell type-dependent roles for secretory autophagy in HCMV infection.
Degree ProgramGraduate College